Genetically encoded Ca2+ sensors hold great promise for the dissection of complex physiology in vivo. The ability to make molecular scale measurements in real time in mammals, and to determine lineage-specific signaling events by genetic specification, provides unprecedented experimental power to determine the complex cell-cell communications that underlie normal organ function, and the dysfunction that attends and is the hallmark of disease. A number of laboratories have developed circularly permutated EGFP-Calmodulin/M13 fusion proteins to understand several complex biological processes in vivo, and these tools have begun to provide a novel window on heart development, heart repair, and endothelial cell signaling. While these studies demonstrate the feasibility of real-time, in vivo imaging at the molecular scale in mammals using genetically encoded Ca2+ indicators, limitations of current molecules prevent their comprehensive exploitation. These limitations include less than optimal signal/noise characteristics, a nonlinear Ca2+ response, the limited spectral range of effective probes, and their non-ratiometric nature. The overall goal is to develop improved genetically encoded Ca2+ sensors (GECIs) through the determination of the structural basis of Ca2+ -dependent fluorescence, the development of multiwavelength indicators that provide the ability to quantify Ca2+ signals in vivo, and the creation of red- shifted GECIs that enable studies of cell-cell signaling in vivo. The effort represents an extension of an ongoing collaboration between the laboratories of Dr. Michael Kotlikoff, who has significant experience with the design and function of GECIs, Dr. Holger Sondermann, who is an expert in protein structure and function, and Dr. Warren Zipfel, a biophysicist with expertise in fluorescence photophysics. These studies will address several significant limitations of current molecules and extend the range of studies for which such molecules will be useful. Emphasis will be placed on the optimization of developed sensors for in vivo performance, as determined by their expression in transgenic mice. PUBLIC HEALTH RELEVANCE: This project will produce novel molecules that can be used in vitro in cell systems and in vivo in animals to determine cellular function in the context of disease or organ repair. The proposal will produce novel proteins that will track the function of cells at the molecular level.